Summary

Over the past few years it has become clear that over half of the mammalian
heart derives from outside the heart field as originally defined. Such a
second heart field, however, has not been described in zebrafish, which could
explain its smaller, two-chambered heart. Instead, zebrafish have a population
of haemangioblasts, which is absent in mammalian embryos, raising the
possibility that these cells represent the evolutionary ancestor of the second
heart field. Here, we show for the first time that the genetic programmes of
these anterior haemangioblasts and the adjacent heart field are co-regulated,
by transcription factors previously associated with heart but not blood or
endothelial development. We demonstrate that gata4, gata5 and
gata6 are essential for anterior haemangioblast specification, and
for subsequent myelopoiesis, acting as early as cloche and upstream
of scl. The requirement for gata4, gata5 and gata6
in myeloid, endothelial and cardiac specification is in the mesoderm, but
these factors also control, from within the endoderm and the yolk syncytial
layer, the migration of the cardiac precursors as they differentiate. This
genetic link between the blood/endothelial and cardiac programmes supports the
notion that this haemangioblast population in zebrafish is an evolutionary
antecedent of the second heart field, and has implications for the
differentiation of haemangioblasts and cardiomyocytes from pluripotent cells,
and for the origins of stem cells in the adult heart.

INTRODUCTION

Of the several candidates for stem cells in the adult heart, only one, in
addition to having the classical stem cell characteristics of
multipotentiality, clonality and self renewal, has also been identified and
traced in living embryos (Martin-Puig et
al., 2008). This cell is identified on the basis of expression of
the homeodomain transcription factor islet 1, and in the embryo these cells
represent the second heart field. In mammals and the chick, the four-chambered
heart is formed from two distinct regions of the embryo: the primary heart
field, which gives rise to the left ventricle and contributes to the atria,
and the second or anterior heart field, which gives rise to the right
ventricle, the outflow tract and also the atria (reviewed by
Laugwitz et al., 2008).
Zebrafish have only two-chambered hearts and no second heart field has
currently been detected. Instead, anterior to the primary heart field,
zebrafish have a population of haemangioblasts not detected in chick or
mammalian embryos.

The anterior lateral plate mesoderm (ALM) in zebrafish is a source of
haematopoietic, endothelial and cardiogenic cells, with the blood and
endothelium coming from the most rostral region and cardiac tissue deriving
from the adjacent more posterior population
(Fig. 1A). The
blood/endothelial precursors in the ALM co-express genes that are later
expressed in either blood or endothelium and have therefore been referred to
as a putative haemangioblast population
(Brown et al., 2000;
Gering et al., 1998;
Thompson et al., 1998). These
ALM haemangioblasts have only a transient existence (between 5 and 10
somites), eventually giving rise to myeloid cells (macrophages and
neutrophils), head endothelium and endocardium
(Herbomel et al., 1999;
Hsu et al., 2001;
Roman and Weinstein, 2000),
whereas the more posterior cardiac precursors differentiate into the muscle of
the two-chambered heart (Stainier and
Fishman, 1992; Stainier et
al., 1993).

Manipulation of anterior haemangioblast regulators suggests that this
programme is antagonistic to the cardiac programme within the ALM
(Gering et al., 2003;
Schoenebeck et al., 2007).
Thus, ectopic expression of blood and endothelial master regulators suppresses
the cardiac programme, whereas knocking them down generates ectopic
cardiomyocytes in the rostral haemangioblast territory. It is tempting to
speculate therefore that this latent cardiac potential found in the anterior
haemangioblast population may have been recruited by amniotes during
evolution, generating the second heart field and a larger, more complex
heart.

Although the anterior haemangioblast and cardiac progenitors express
predominantly distinct sets of genes, a few are expressed in both territories;
for example, gata4, gata5 and gata6
(Reifers et al., 2000;
Reiter et al., 1999;
Reiter et al., 2001;
Schoenebeck et al., 2007)
(this study). Clearly, if the anterior haemangioblast population is the
evolutionary precursor of the second heart field, one would expect that they
would be under common genetic control prior to the separation of the two
programmes. Jointly expressed transcriptional regulators such as gata4,
gata5 and gata6 are therefore candidates for such common genetic
control. In vertebrates, GATA factors are traditionally described as belonging
to two subfamilies: those predominantly expressed and functioning in
haematopoiesis and ectodermal patterning (gata1, gata2 and
gata3), and those playing a role in cardiac and endodermal formation
(gata4, gata5 and gata6)
(Molkentin, 2000;
Patient and McGhee, 2002). In
zebrafish, gata4, gata5 and gata6 have indeed already been
shown to be required for normal cardiogenesis and the formation of heart
precursors (Holtzinger and Evans,
2007; Peterkin et al.,
2003; Peterkin et al.,
2007). To determine whether the anterior haemangioblast population
is under common genetic control with the heart field, we used morpholinos to
knock down gata4, gata5 and gata6, and show for the first
time that they are crucial for anterior haemangioblast formation and
subsequent myelopoiesis. This requirement is within the mesoderm, although we
also show that gata5 and gata6 are required in the yolk
syncytial layer (YSL) and the endoderm for the correct migration of cardiac
precursors. The ablation of both cardiac and haemangioblast programmes within
the ALM suggests that these GATA factors lie at the top of a genetic cascade
that is initially common to both of these two lineages. This is confirmed by
the continued expression of gata4, gata5 and gata6 in
scl morphants and cloche mutants, suggesting that these GATA
factors lie upstream of, or parallel to, these well-described blood and
endothelial regulatory factors. These data genetically link the anterior
haemangioblast and cardiac fields, and are consistent with the former being
the evolutionary ancestor of the latter.

MATERIALS AND METHODS

In situ hybridisation of zebrafish embryos

Wild-type and cloche [Clom39]
(Stainier, 2001) zebrafish
were bred, maintained, and embryos raised and staged using standard conditions
(Westerfield, 1993). In situ
hybridisations on zebrafish embryos were carried out as previously described
(Jowett, 2001). All RNA probes
used were labelled with digoxigenin (DIG) and detection of the
antibody-alkaline phosphatase was performed using BM purple (Roche). EST
clones for ets1 and erg1 were obtained from ImaGenes, clone
ID 7051215 and 6966926, respectively. The following forward
(5′-ATGATGGATAGCCGGATCCTCG-3′) and reverse
(5′-GTCCATGTCTACATCCTCTCC-3′) primers were used to amplify
uncx4.1 from cDNA using standard PCR protocols. The PCR fragment was
cloned into the pGEM T-Easy vector (Promega) and confirmed by sequencing. To
make an in situ hybridisation probe, uncx4.1 was linearised with
Spe1 and transcribed with T7. Details of all other probes have been
described previously (Patterson et al.,
2005; Peterkin et al.,
2003; Peterkin et al.,
2007).

Morpholino and mRNA injections

The gata5 and gata6 antisense morpholinos (mo) were
designed and manufactured by Gene Tools and sequences have been previously
described (Peterkin et al.,
2003; Peterkin et al.,
2007). Combinatorial injections were mixed at a ratio of 1:5
gata6mo:gata5mo and titrated to an appropriate level to
avoid non-toxic effects: between 1.25-1.50 ng of gata6mo and
6.25-8.50 ng gata5mo. The sequences of other morpholinos used have
been described previously: scl splice mo
(Patterson et al., 2005);
pu1 mo (Rhodes et al.,
2005); and casanova mo
(Sakaguchi et al., 2001).
scl, pu1 and cas morpholinos were titrated to around amounts
previously described; final quantities of 6.5 ng, 15 ng and 10 ng,
respectively, were used. YSL injections were performed at the 1000-cell stage
using a standard fluorescent control morpholino (Gene Tools) as a lineage
tracer. scl and lmo2 mRNAs were synthesised and injected as
previously described (Gering et al.,
2003), etsrp (etv2 - Zebrafish Information
Network) mRNA was synthesised and injected as described
(Pham et al., 2007).

RESULTS

gata4, gata5 and gata6 are required for
the formation of the anterior haemangioblast

gata4, gata5 and gata6 are expressed in the ALM of
zebrafish embryos during early somitogenesis, in the region that gives rise to
primitive myeloid cells, head endothelium and endocardium
(Herbomel et al., 1999;
Reifers et al., 2000;
Reiter et al., 1999;
Reiter et al., 2001;
Schoenebeck et al., 2007)
(Fig. 1A, `anterior
haemangioblasts'). Initially forming bilateral stripes either side of the
embryo, the precursors migrate to a position just anterior to the heart cone
as they differentiate, and the myeloid derivatives then disperse throughout
the whole embryo, expressing markers such as l-plastin
(lymphocyte cytosolic plastin 1 - Zebrafish Information Network) and
mpx (myeloperoxidase) in monocytes/macrophages and
granulocytes, respectively. To determine whether gata4, gata5 or
gata6 play any role in myeloid development, we used morpholinos to
combinatorially knock down gata5 and gata6. We have
previously shown that these double morphants are essentially a triple
knockdown, as gata4 is no longer expressed, which was reconfirmed
here (see Fig. S1A in the supplementary material)
(Peterkin et al., 2007). In
gata5 and gata6 morphants at 24 hours post-fertilization
(hpf), both l-plastin and mpx were severely downregulated
(Fig. 1B). Loss of the
transcription factor ikaros, associated with less-differentiated
cells, was also seen in the head region of morphants
(Fig. 1B), suggesting that the
defect might be prior to terminal differentiation. Probing earlier for
cmyb and ikaros expression confirmed this, as expression was
absent in morphants close to the onset of their expression at 10 somites
(Fig. 1C). Expression of the
key myeloid regulator pu1 (spi1 - Zebrafish Information
Network) was also ablated at this time
(Fig. 1C). These data show that
gata4, gata5 and gata6 are essential for the specification
of myeloid cells during development.

Recent studies have identified both redundant and non-redundant
contributions from gata4, gata5 and gata6 during development
(Holtzinger and Evans, 2005;
Holtzinger and Evans, 2007;
Peterkin et al., 2007). To
address this issue in the myeloid population, gata4, gata5 or
gata6 morpholinos were injected individually. The expression of both
l-plastin and mpx was much less severely downregulated in
all three individual morphants than in morphants in which all three were lost
together (compare Fig. S1B in the supplementary material with
Fig. 1B), which suggests that
their activities are additive. Therefore, for the rest of the experiments
described here, gata5 and gata6 morpholinos were
co-injected, creating a triple knockdown.

The loss of the earliest myeloid regulators at 10 somites led us to explore
the extent to which the entire anterior haemangioblast programme is disrupted.
draculin and gata2 are the first blood-associated genes to
be expressed in the ALM between 1 and 2 somites
(Patterson et al., 2005), and
expression of both genes was substantially reduced in gata5 and
gata6 morphants (Fig.
1D, red arrowheads). Likewise, expression of scl, fli1,
lmo2 and etsrp, which are expressed in and required for
haemangioblast formation, were severely downregulated
(Gering et al., 1998;
Liu et al., 2008;
Patterson et al., 2007;
Patterson et al., 2005;
Sumanas and Lin, 2006)
(Fig. 1D). Similar
downregulation was observed for erg1, ets1 and hhex, whose
later expression is in endothelial cells
(Liao et al., 2000;
Liu and Patient, 2008;
Sumanas and Lin, 2006)
(Fig. 1D, red arrowheads). By
contrast, expression of all of these genes in the posterior lateral mesoderm
(PLM), which gives rise to erythroid cells and the major vessels, was
unaltered (see Fig. S2 in the supplementary material). Staging of these early
embryos was confirmed by counting somite numbers after staining for
uncx4.1 expression (Kawakami et
al., 2005) (Fig.
1D, white brackets; data not shown). These data demonstrate a very
early role for gata4, gata5 and gata6 in the specification
of anterior haemangioblasts.

Rescue experiments were carried out to validate morpholino specificity. The
severe downregulation of fli1, etsrp, scl and pu1 seen in
morphants was rescued by the injection of gata5 and gata6
mRNA (Fig. 2). Overexpression
studies of GATA factors have proven to be difficult because of their strong
phenotypes (Haworth et al.,
2008; Weber et al.,
2000); however, low levels of gata5 and gata6
(25 pg) injected into wild-type embryos produced relatively normal embryos
morphologically, and the blood-associated genes pu1 and scl
were ectopically expressed (Fig.
2). By contrast, very little if any ectopic or increased
expression of vascular genes was observed
(Fig. 2). A few ectopic patches
of cells expressing fli1 were detected, whereas etsrp
expression was seen only within the normal bilateral ALM stripes. Thus,
although gata5 and gata6 are necessary for all
haemangioblast gene expression in the ALM, they are sufficient only for
myeloid and not vascular gene expression.

gata4, gata5 and gata6 are essential for
anterior haemangioblast specification. (A) gata4, gata5
and gata6 are expressed in overlapping yet distinct domains
throughout the anterior lateral plate mesoderm (ALM). (B) Expression of
l-plastin, mpx and ikaros, genes associated with
myelopoiesis, was absent in gata5+gata6 morphants at 24 hpf.
(C) Analysis of earlier regulatory myeloid gene expression on knock
down of gata5 and gata6 shows ablation of cmyb,
ikaros and pu1 at 10 somites (red arrowheads). (B,C) The number
of embryos exhibiting this phenotype shown at the bottom right-hand corner of
each panel. (D) Expression of haemangioblast-associated genes close to
the onset of their expression was downregulated in gata5+gata6
morphants (marked by red arrowheads where necessary). uncx4.1 was
used as a somite marker to stage the embryos (white brackets).
n=40-100 for each gene analysed, the pictures shown depict >95% of
the embryos. Views are: (A) flatmounted, anterior to the top; (B) lateral,
anterior to left, except for ikaros, which is an anterior view; (C)
dorsal, anterior to the top; (D) anterior-dorsal.

gata5 and gata6 play a migratory role in the
YSL

gata5 and gata6 are expressed in the endoderm and the
yolk syncytial layer (YSL), as well as in the ALM, in zebrafish embryos
(Kikuchi et al., 2001;
Reiter et al., 1999;
Rodaway et al., 1999) (A.G.,
T.P. and R.P., unpublished). The YSL appears to be crucial for migration of
cardiac precursors to the midline, with YSL defects giving rise to cardia
bifida (Sakaguchi et al.,
2006). Cardia bifida is also seen in gata5 and
gata6 morphants (Peterkin et al.,
2007), suggesting that these factors may be acting in the YSL. We
therefore wished to determine whether the activity affecting anterior
haemangioblast programming is located in the YSL or the ALM. To deplete
gata5 and gata6 in the YSL, 1000-cell stage embryos were
injected into the YSL with gata5 and gata6 morpholinos,
along with a fluorescent morpholino as a tracer. At this stage of development,
the YSL gap junctions are complete and no longer open to the embryo itself
(Chen and Kimelman, 2000;
Kimmel and Law, 1985). Embryos
fluorescent only in the YSL were selected at two different stages as embryos
lacking gata5 and gata6 in the YSL alone
(Fig. 3A, +YSL). Embryos with
no fluorescence (-YSL) were collected as injection controls, expressing
gata5 and gata6 at wild-type levels. To confirm the
efficiency of the morpholinos, a batch of embryos was injected at the one-cell
stage, giving rise to fluorescence, and therefore gata5 and
gata6 inhibition, throughout the embryo (+embryo). Cardiac and
myeloid gene expression was monitored.

As expected, the levels of expression of the cardiac markers cmlc2
and nkx2.5 were normal in the injection controls (-YSL), as were the
locations of the expressing cells (Fig.
3A). However, in the embryos in which gata5 and
gata6 were absent in the YSL alone (+YSL), 100% of the embryos showed
cardia bifida, although the levels of expression remained normal
(Fig. 3A). In the embryos used
as a control for morpholino efficiency (+embryo), cmlc2 and
nkx2.5 expression was absent, as reported previously
(Peterkin et al., 2007).
Importantly though, the expression of the myeloid marker l-plastin
also remained unchanged in + and -YSL embryos, but was ablated when
gata5 and gata6 MOs were injected throughout the embryo
(+embryo; see Fig. 3A). Thus,
gata5 and gata6 do not appear to be required in the YSL for
myelopoiesis, but rather in the embryo proper. These experiments also show
that, whilst expression of gata5 and gata6 is required in
the YSL for cardiac migration, it is not required there for heart
specification or differentiation.

gata5 and gata6 rescue and gain-of-function
experiments confirm necessity and identify sufficiency for myelopoiesis.
Injection of exogenous gata5 and gata6 mRNA (g5+6RNA) into
wild-type embryos did not induce fli1 or etsrp, whereas
scl and pu1 were ectopically expressed. Rescue of all four
genes was seen in gata5 and gata6 morphants co-injected with
gata5 and gata6 mRNA (g5+6mo + g5+6RNA) when compared with
gata5 and gata6 morphants alone (g5+6mo). Views are
anterior-dorsal.

gata5 and gata6 play a migratory role in the
endoderm

In zebrafish, endoderm is not thought to be required for the specification
and differentiation of heart precursors, but it is required for the correct
migration of cardiac progenitors to the midline
(Alexander et al., 1999;
Kikuchi et al., 2001;
Schier et al., 1997;
Trinh and Stainier, 2004).
However, its role in myeloid development has not been assessed, and therefore
the role of gata4, gata5 and gata6 in the embryo proper
(shown above) could in principle be in the endoderm. In casanova
(cas) mutants, which have no endoderm, gata4, gata5 and
gata6 expression is still observed in the mesoderm
(Alexander et al., 1999;
Kikuchi et al., 2001), thus
the roles of gata4, gata5 and gata6 in the endoderm and
mesoderm should be distinguishable. When wild-type embryos were injected with
a cas morpholino, endodermal gata4, gata5 and gata6
expression was lost at 27 hpf, as has been described for cas mutants
(Alexander et al., 1999) (see
Fig. S3A in the supplementary material, red arrowheads), whereas the
expression of these GATA factors in the cardiac mesoderm was maintained,
albeit in a pattern similar to that seen in cardia bifida, as is seen for
cmlc2 (see Fig. S3A in the supplementary material, black arrowheads).
Importantly, expression of l-plastin and mpx at this time
was not altered, suggesting that endoderm, and therefore gata4, gata5
and gata6 expression within the endoderm, is not required for
myelopoiesis at 27 hpf (Fig.
3B).

Expression of gata4, gata5 and gata6 in the ALM at 5
somites was unaffected in cas morphants (see Fig. S3B in the
supplementary material). Furthermore, expression of pu1 at 5 somites
and runx1, ikaros and cmyb at 10 somites was also unaffected
(Fig. 3C). As a control for
morpholino activity (Dickmeis et al.,
2001), expression of the endodermal marker sox17 was lost
by 50-90% epiboly (data not shown). We therefore conclude that gata4,
gata5 and gata6 are required in the mesoderm for anterior
haemangioblast, as well as cardiac, specification, whereas gata5 and
gata6 are required in the endoderm and the YSL only for the migration
of cardiac precursors.

Epistatic relationships between gata4, gata5 and
gata6 and haemangioblast genes

Having established that the anterior haemangioblast requirement for
gata4, gata5 and gata6 is in the mesoderm itself, we wanted
to determine their position in the genetic hierarchy. pu1 expression
is lost in gata5 and gata6 morphants but, although this
could explain the absence of myelopoiesis, a role for pu1 in
haemangioblast formation or endothelial development has not been established
(Rhodes et al., 2005;
Su et al., 2007). However,
analysis of pu1 morphants showed that pu1 is required only
for myeloid development and not at all for the endothelial programme (see Fig.
S4B,C in the supplementary material). We conclude that the loss of
pu1 in gata5 and gata6 morphants
(Fig. 1C) and the continued
expression of gata4, gata5 and gata6 in pu1
morphants (see Fig. S4B in the supplementary material), together with the more
widespread defects observed in haemangioblast formation in gata5 and
gata6 morphants, places gata5 and gata6 upstream of
pu1. We also note that, even though pu1 was absent in the
ALM of gata5 and gata6 morphants, ectopic globin
and gata1 was never seen there, in contrast to pu1 morphants
(Fig. S4B in the supplementary material; data not shown)
(Rhodes et al., 2005). Thus,
in the absence of gata4, gata5 and gata6, the ALM is unable
to form either myeloid or erythroid blood, which is consistent with a position
higher up the hierarchy than pu 1.

scl has been implicated in haemangioblast formation in both the
ALM and the PLM of zebrafish embryos
(Bussmann et al., 2007;
Gering et al., 2003;
Patterson et al., 2005). In
the ALM, expression of the blood genes pu1, cmyb, runx1 and
ikaros is disrupted, along with expression of the endothelial genes
flt4 and hhex, when scl is lost, placing it towards
the top of the anterior haemangioblast hierarchy
(Patterson et al., 2005). To
establish the relationship between scl and gata4, gata5 and
gata6, the expression of GATA factors was investigated in
scl morphants. Embryos were injected with the scl
morpholinos previously described
(Patterson et al., 2005).
Continued expression of gata4, gata5 and gata6 was seen in
scl morphants from 7 somites (Fig.
4A) to around 15 somites (data not shown). As a control for
morpholino functionality, pu1 was lost in scl morphants, as
has been shown previously (Patterson et
al., 2005) (Fig.
4A). Thus, as scl is lost in GATA morphants
(Fig. 1D), and gata4,
gata5 and gata6 expression is maintained in scl
morphants (Fig. 4A), gata4,
gata5 and gata6 emerge as upstream regulators of scl in
the ALM.

cloche mutants lack vascular and haematopoietic tissues, including
myeloid cells (Liao et al.,
1997; Rhodes et al.,
2005; Thompson et al.,
1998), and gata4, gata5 and gata6 lie upstream
of scl and pu1, both of which can partially rescue
myelopoeisis in cloche embryos
(Liao et al., 1997;
Rhodes et al., 2005).
Determining the expression of cloche has not been possible, because
of uncertain identification and low expression of the only candidate
(Xiong et al., 2008), so we
could not monitor cloche expression in gata5 and
gata6 morphants. We therefore assessed the expression of
gata4, gata5 and gata6 in cloche embryos.
To control for both the number of somites and to identify cloche
embryos from their wild-type siblings, triple in situ hybridisation was
performed (Fig. 4B,C).
gata4, gata5 and gata6 expression was monitored in the ALM
(red arrowheads), and in the same embryos uncx4.1 staining of early
somites was used as a timing control (white brackets), while the loss of
gata1 expression in the PLM was used to identify cloche
embryos (Stainier et al.,
1995). Expression levels of all three GATA factors remained
unchanged in cloche embryos (Fig.
4B), even though pu1 expression in the ALM was lost, as
shown previously (Lieschke et al.,
2002). Thus, gata4, gata5 and gata6 lie upstream
or parallel to cloche in the ALM.

Depletion of gata5 and gata6 from the YSL causes
cardia bifida but does not affect myelopoiesis. (A) The YSL
expression of gata5 and gata6 was depleted by injection of
gata5+gata6 morpholinos into the YSL at the 1000-cell stage. To trace
the correctly targeted embryos, a fluorescent control morpholino was
co-injected. Embryos positive for fluorescence in the YSL alone (+YSL) were
selected at several time points and harvested as YSL
gata5+gata6-depleted embryos. The embryos showing no fluorescence
(-YSL) were collected as negative injection controls and should express
wild-type levels of gata5 and gata6. To ensure the
efficiency of the morpholinos, positive control embryos were injected with
gata5+gata6 morpholinos at the one-cell stage and were fluorescent
throughout the embryo (+embryos). To assess heart formation, cmlc2
and nkx2.5 expression was analysed. Depletion of gata5 and
gata6 in the YSL (+YSL) showed normal levels of expression of both
cmlc2 and nkx2.5, indicating that specification occurs
normally in these embryos. However, cardia bifida was observed in the +YSL
embryos, demonstrating that gata5 and gata6 are required in
the YSL for the correct migration of the cardiac precursors to the midline. By
contrast, l-plastin expression was the same as in the wild-type
embryos, indicating that gata5 and gata6 expression in the
YSL is dispensable for myelopoiesis. Embryos injected at the one-cell stage
(+embryos) showed complete absence of cmlc2, nkx2.5 and
l-plastin expression, thereby validating morpholino effectiveness.
Views are anterior. For each gene and type (±YSL, +embryo) analysed,
n=28-38, and the images shown depict the findings for >95% of the
embryos. (B,C) Loss of endoderm in casanova morphant embryos
but no defects in myelopoiesis. To establish whether endoderm plays a role in
myelopoiesis, endodermless embryos were created by injection of
casanova morpholinos. Casanova morphants (casmo)
were assessed for endoderm formation and myelopoiesis. Myelopoiesis was not
affected in cas morphants as l-plastin and mpx
remained unaffected (B). Gene expression in the ALM of cas morphants
was examined at 5 and 10 somites (C). The formation of myeloid precursors
occurred as normal in cas morphants. Expression of pu1 at 5
somites, and runx1, ikaros and cmyb at 10 somites, was
unaffected in cas morphants. Views are anterior-dorsal. For each gene
analysed n=38-67, and the images shown depict the findings for>
95% of the embryos.

To confirm the hierarchical relationships between gata4, gata5 and
gata6 and the other haemangioblast-associated genes, we tried to
rescue myeloid and vascular gene expression in gata5 and
gata6 morphants. Previous studies have shown that co-injection of
scl and lmo2 can strongly induce ectopic haemangioblast gene
expression, although myeloid gene expression was not expanded and the cells
appeared disorganised (Gering et al.,
2003). By contrast, etsrp overexpression induced ectopic
vasculogenesis and myelopoiesis (Sumanas
et al., 2008). Overexpression of scl/lmo2 or
estrp mRNAs in wild-type embryos behaved as described above (data not
shown). However, injection of scl/lmo2 mRNAs into gata5 and
gata6 morphants, while still able to strongly induce fli1
(as in wild-type embryos), was also able to rescue pu1 expression,
albeit in a disorganised fashion (Fig.
5A). By contrast, although etsrp was able to induce
fli1 expression in the gata5 and gata6 morphants,
it could not rescue pu1 expression
(Fig. 5A). Together with the
gata5 and gata6 overexpression data above, and with recent
evidence that etsrp is required for the myeloid programme
(Liu and Patient, 2008;
Sumanas et al., 2008), these
data confirm differential roles for gata5 and gata6 and
etsrp in endothelial versus myeloid development
(Fig. 5B). gata5 and
gata6 are necessary and sufficient for at least pu1
expression, probably via the induction of scl, whereas they are
necessary but not sufficient for etsrp and fli1 expression,
suggesting that an additional factor is required (X in
Fig. 5B). The failure of
etsrp to rescue pu1 expression while inducing fli1
expression in gata5 and gata6 morphants, suggests that
etsrp is dependent on gata5 and gata6 for its
downstream activity in myeloid but not endothelial development. Similarly, the
ability of gata5 and gata6 to induce other endothelial
markers may depend on etsrp.

The fate of gata4-, gata5- and
gata6-depleted mesoderm

The data presented here and in previous reports show that gata5
and gata6 are required for the specification of the cardiac and
haemangioblast programmes in the ALM
(Peterkin et al., 2003;
Peterkin et al., 2007;
Reiter et al., 1999). What
happens to these cells in the absence of gata4, gata5 and
gata6? We showed previously that expression of nkx2.7 is
unaffected in the ALM at 5 somites when haemangioblast- and cardiac-associated
genes are already lost in gata5 and gata6 morphants, and
that no increase in apoptosis was observed at 10 somites
(Peterkin et al., 2007). From
these data we can conclude that mesodermal cells are still present and have
correctly migrated during gastrulation, but that they are unable to
differentiate into either the cardiac or haemangioblast lineages. Additional
mesodermal markers expressed in the ALM in the absence of the haemangioblast
and cardiac programmes are not currently available, so we monitored adjacent
tissues to determine whether they are expanded in the absence of
gata5 and gata6. Fin buds are one candidate; however, they
are dependent on tbx5 (Ahn et al.,
2002; Garrity et al.,
2002) and tbx5 is absent in gata5 and
gata6 morphants at 10 somites
(Peterkin et al., 2007).
Consistent with this, a loss of fin buds was evident at 27 hpf, as revealed by
the loss of blimp and shh expression
(Fig. 6A, red arrows).
blimp expression in the pharyngeal endoderm was slightly reduced but
still present (Fig. 6A, black
arrows). wt1 is expressed in the pronephros just posterior to the
heart field (Drummond et al.,
1998; Serluca and Fishman,
2001), and this expression was unaffected in gata5 and
gata6 morphants (Fig.
6B). Thus, in the absence of gata5 and gata6,
the haemangioblast, cardiac and fin bud programmes are not induced, but
adjacent tissues are not expanded and mesodermal cells expressing
nkx2.7 are still present in the ALM.

Initiation of gata4, gata5 and gata6
expression is independent of scl and cloche. (A)
Expression of the GATA factors was analysed in scl morphants.
gata4, gata5 and gata6 were expressed as in the wild-type
embryos in the ALM (red arrowheads), placing them upstream of scl.
Loss of pu1 was used as a control for scl morpholino
efficacy. (B) The expression of gata4, gata5 and
gata6 in the ALM was unchanged (red arrowheads) in cloche
embryos placing the GATA factors parallel to or upstream of cloche.
(C) cloche embryos were identified by the absence of
gata1 expression (red arrowheads) in the PLM. Pu1
downregulation was an additional control (B). The expression of
uncx4.1 was used as a somitic counter to ensure correct staging of
the embryos (white brackets). Views are anterior-dorsal. For each gene
analysed, n=28-42, and the images shown depict the findings for>
95% of the embryos identified as cloche and scl morphant
embryos.

Interaction of gata5 and gata6 with scl/lmo2
and etsrp. (A) Scl/lmo2 mRNA can rescue both
blood (pu1), and endothelium (fli1 and etsrp)
development in gata5 and gata6 morphants at the 5 somite
stage. By contrast, etsrp overexpression can rescue fli1 but
not pu1 expression in gata5 and gata6 morphants.
All views are anterior. N.D., not determined. (B) Proposed model for
gata5 and gata6 function within the anterior haemangioblast.
gata5 and gata6 are required for etsrp and
scl expression in the haemangioblast. gata5 and
gata6 overexpression can induce scl and pu1
expression, which drives the myeloid lineage, whereas endothelium cannot be
induced by gata5 and gata6 mRNA (see
Fig. 2). Furthermore, the head
vasculature eventually recovers (see Fig. S4A in the supplementary material),
suggesting the existence of an unknown GATA-independent signal (X).

Cellular hierarchy of anterior haemangioblast and heart formation.gata4, gata5 and gata6 are required for the specification of
the anterior haemangioblast and cardiac precursors from the anterior lateral
plate mesoderm (double red break). gata4, gata5 and gata6
lie at the apex of the hierarchy, above cloche (double red break) and
scl. Previous studies show that loss of the haemangioblast programme
causes the expansion of cardiac progenitors
(Schoenebeck et al., 2007),
suggesting an antagonistic relationship between these two programmes (barred
solid and dashed lines). Whether the cardiac precursors can antagonise the
anterior haemangioblast population has yet to be demonstrated (dashed line).
Data presented here also show a requirement for gata5 and
gata6 in the endoderm and the YSL for the correct migration of
cardiac precursors towards the midline.

What happens to the nkx2.7-expressing cells? Expression of myeloid
genes such as runx1, ikaros, draculin, l-plastin and mpx in
ALM-derived cells was never seen in gata5 and gata6
morphants (Fig. 1; data not
shown), whereas endothelial differentiation appeared to have recovered
completely by 22 hpf (see Fig. S4A in the supplementary material). This
recovery is consistent with previous data
(Holtzinger and Evans, 2007),
which have shown that expression of lmo2 appeared normal in
gata5 and gata6 morphants around 12 somites, a time at which
we saw recovery of expression of several endothelial genes, including
lmo2. Thus, it appears that some of the ALM cells in morphants are
still able to contribute to head vasculature.

DISCUSSION

gata4, gata5 and gata6 lie at the
hierarchical apex of blood and cardiac specification

Data presented here demonstrate for the first time a crucial role for
gata4, gata5 and gata6 in haemangioblast as well as cardiac
precursor specification. Although Gata4 and Gata6 have been detected in mouse
embryos in and around the blood islands and the allantois, another tissue
associated with blood and endothelial development, there has hitherto been no
evidence for a cell-autonomous role
(Bielinska et al., 1996;
Caprioli et al., 2001;
Dumon et al., 2006;
Nemer and Nemer, 2003).
Gata5 expression has been observed to increase in embryonic stem (ES)
cells differentiated towards a haematopoietic fate, but no functional
consequences have been demonstrated (Baird
et al., 2001). Here, we show for the first time that expression of
these `cardiac' GATA factors is required in the lateral mesoderm for the
development of myeloid lineages and the initial programming of their
associated endothelial cells.

The presence of gata4, gata5 and gata6 in the endoderm
and YSL, as well as in the cardiac and haemangioblastic mesoderm, raised
issues concerning the cell autonomy of the role described here. We demonstrate
that gata5 and gata6 are indeed crucial in both the YSL and
the endoderm for migration of cardiac precursors to the midline, but that they
are dispensable there for the specification of both the heart tissue and the
haemangioblasts. Thus, the requirement for the GATA factors in specification
must reside within the mesoderm. Precedent for inductive interactions between
endoderm and blood/endothelial programming comes from experiments performed in
mouse and chick (Belaoussoff et al.,
1998; Bielinska et al.,
1996; Kessel and Fabian,
1987). By contrast, we found that loss of endoderm in
casanova morphants, including loss of gata4, gata5
and gata6 expression there, had no effect on haemangioblast
specification or myelopoiesis. Differences between zebrafish and mouse/chick
may in part reflect the different origins of the endoderm: whereas in chick
and mouse, yolk sac haematopoiesis and vasculogenesis occur adjacent to the
visceral endoderm, in zebrafish the ALM is adjacent to the definitive
endoderm.

The mutated gene in the zebrafish cloche mutant is thought to be
close to the hierarchical apex of blood and endothelial development
(Liao et al., 1997;
Rhodes et al., 2005;
Thompson et al., 1998).
Consistent with this notion, the mouse homologue of lycat, a gene
cloned from the cloche genetic interval, is essential for blood and
endothelial specification in ES cells
(Wang et al., 2007). We have
now shown that gata4, gata5 and gata6 are required for both
haemangioblast and cardiac specification, and that expression of gata4,
gata5 and gata6 is unaffected in cloche mutants,
placing these GATA genes not only upstream of or parallel to
cloche/lycat in the haemangioblast lineage, but also at the
apex of the genetic hierarchy common to both the cardiac and haemangioblast
programmes (Fig. 7). Based on
their early expression patterns (Rodaway
and Patient, 2001), we hypothesise that gata5 and
gata6 are required very early in the mesendoderm, allowing it to
respond to both blood- and cardiac-inducing signals.

Although the requirement for gata4, gata5 and gata6 for
blood development from the anterior haemangioblast is absolute, there appears
to be a GATA-independent pathway that is able to rescue endothelial
development after GATA expression has ceased. Thus, whereas expression of the
myeloid genes in the anterior (pu1, runx1, cmyb, l-plastin and
mpx) was never seen in gata5 and gata6 morphants,
expression of genes associated with endothelial development (such as fli1,
etsrp1, ets1 and hhex), along with haemangioblast genes
(scl and lmo2), in their later head endothelial mode, began
to recover from around 12 somites and appeared normal by 15 somites, resulting
in an apparently normal circulatory system (see Fig. S4A in the supplementary
material; data not shown). Thus, it appears that a recovery pathway is
available for endothelial development from the ALM but not for blood
(Fig. 5B, labelled X). As
cloche embryos show a severe downregulation of endothelium throughout
development, it is likely that the recovery of head endothelium in GATA
morphants is cloche dependent. Thus, even though gata5 and
gata6 are required together with cloche for the initiation
of the haemangioblast programme, they are apparently not required for
maintenance of the endothelial programme in the haemangioblast
derivatives.

gata4, gata5 and gata6 and the origins of
the second heart field and cardiac stem cells

Attractive candidate stem cells in the adult mouse heart are the islet
1-positive population also found in developing embryos (reviewed by
Laugwitz et al., 2008). These
cells constitute the second heart field in the mammalian embryo. Although the
absence of a second heart field in zebrafish embryos may explain the smaller
two-chambered heart produced, the presence of a haemangioblast population
anterior to the primary heart field, and its absence in mouse embryos, raises
the possibility that this haemangioblast population represents the
evolutionary precursor of the mammalian second heart field. Consistent with
such a notion, we have found that the two populations do indeed have common
genetic control, depending absolutely in both cases on gata4, gata5
and gata6. Interestingly, using the marker islet-1, a second
heart field has recently been reported for the amphibian Xenopus
laevis (Brade et al.,
2007), which has a three-chambered heart and an anterior
haemangioblast population (Walmsley et
al., 2002), raising the possibility that Amphibia represent an
intermediate evolutionary state between fish and amniotes.

Recently, cardiac gene expression has been detected in the anterior
haemangioblast population in cloche embryos and in
scl/etsrp morphants, suggesting that, normally, the
blood/endothelial programme there might be responsible for the suppression of
the cardiac programme (Schoenebeck et al.,
2007). Consistent with this, overexpression of scl, with
either etsrp or lmo2, ablates heart formation
(Gering et al., 2003;
Schoenebeck et al., 2007).
These observations suggest that the blood and cardiac programmes in the ALM
are antagonistic. It will be interesting to determine how this antagonism was
resolved in favour of the cardiac programme during evolution.

Identifying and characterising stem cells in the adult heart is likely to
have implications for treatment of heart disease. Islet 1-positive cells have
several of the necessary credentials and are thought to derive from the second
heart field. Clearly a better understanding of their genetic programme will
facilitate their future manipulation and also their derivation from
pluripotent cells. Consistent with the common genetic programme indicated
here, cardiomyocytes have recently been obtained from ES cell derivatives
expressing the VEGF receptor, flk-1, which is classically associated
with haemangioblast development (Kattman
et al., 2006). Interestingly, haemangioblasts differentiate from
ES cells before cardiomyocytes and in less time than it takes to make
haemangioblasts in a mouse embryo (Huber
et al., 2004). In zebrafish and more obviously Xenopus
embryos, the anterior haemangioblast programme develops earlier than the
posterior blood and endothelial programme
(Walmsley et al., 2008),
raising the intriguing possibility that the haemangioblasts derived from mouse
ES cells are expressing the ancestral programme.

Taken together, our work identifies a close genetic relationship between
anterior haemangioblasts and cardiac precursors in zebrafish. We reveal a
co-dependence of these populations on gata4, gata5 and
gata6, placing these genes at the apex of the genetic regulatory
hierarchy specifying these two anterior lateral mesoderm derivatives. This
common genetic control is consistent with the postulated conversion of one to
the other during evolution.

Supplementary material

Acknowledgments

We would like to thank Shuo Lin for probes, Didier Stainier for the
cloche mutants and Brant Weinstein for the etsrp construct.
EST clones were obtained from ImaGenes. We are grateful to the staff of the
Biomedical Services Unit for aquatic support. We appreciate the helpful
comments from Maggie Walmsley, Filipa Simoes and the reviewers, and thank
Philip Pinheiro for his help.

Footnotes

This work was supported by the British Heart
Foundation and MRC. Deposited
in PMC for release after 6 months.

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